Phosphatidylinositol 4,5‐bisphosphate degradation inhibits the Na + /bicarbonate cotransporter NBCe1‐B and ‐C variants expressed in Xenopus oocytes

Key points We previously reported that the phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) directly stimulates heterologously expressed electrogenic Na + /bicarbonate cotransporter NBCe1‐A in an excised macropatch from the Xenopus oocyte, and indirectly stimulates NBCe1‐B and ‐C in the intact oocyte primarily through inositol 1,4,5‐trisphosphate/Ca 2+ . In the current study, we expand on a previous observation that PIP 2 may also directly stimulate NBCe1 in the intact oocyte. In this study on oocytes, we co‐expressed either NBCe1‐B or ‐C and a voltage‐sensitive phosphatase (VSP), which depletes PIP 2 without changing inositol 1,4,5‐trisphosphate, and monitored NBCe1‐mediated currents with the two‐electrode voltage‐clamp technique or pH i changes using V m /pH‐sensitive microelectrodes. Activating VSP inhibited NBCe1‐B and ‐C outward currents and NBCe1‐mediated pH i increases, and changes in NBCe1 activity paralleled changes in surface PIP 2 . This study is a quantitative assessment of PIP 2 itself as a regulator of NBCe1‐B and ‐C in the intact cell, and represents the first use of VSP to characterize the PIP 2 sensitivity of a transporter. These data combined with our previous work demonstrate that NBCe1‐B and ‐C are regulated by two PIP 2 ‐mediated signalling pathways. Specifically, a decrease in PIP 2 per se can inhibit NBCe1, whereas hydrolysis of PIP 2 to inositol 1,4,5‐trisphosphate/Ca 2+ can stimulate the transporter.Abstract The electrogenic Na + /bicarbonate cotransporter (NBCe1) of the Slc4 gene family is a powerful regulator of intracellular pH (pH i ) and extracellular pH (pH o ), and contributes to solute reabsorption and secretion in many epithelia. Using Xenopus laevis oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) directly stimulates NBCe1‐A in an excised macropatch, and indirectly stimulates NBCe1‐B and ‐C in the intact oocyte primarily through inositol 1,4,5‐trisphosphate (InsP 3 )/Ca 2+ . In the current study, we used the two‐electrode voltage‐clamp technique alone or in combination with pH/voltage‐sensitive microelectrodes or confocal fluorescence imaging of plasma membrane PIP 2 to characterize the PIP 2 sensitivity of NBCe1‐B and ‐C in whole oocytes by co‐expressing a voltage‐sensitive phosphatase (VSP) that decreases PIP 2 and bypasses the InsP 3 /Ca 2+ pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1‐B/C current, consistent with an initial rapid depolarization‐induced NBCe1 activation, and then a subsequent slower VSP‐mediated NBCe1 inhibition. Upon repolarization, the NBCe1 current decreased, and then slowly recovered with an exponential time course that paralleled PIP 2 resynthesis as measured with a PIP 2 ‐sensitive fluorophore and confocal imaging. A subthreshold depolarization that minimally activated VSP caused a more sustained increase in NBCe1 current, and did not lead to an exponential current recovery following repolarization. Similar results were obtained with oocytes expressing a catalytically dead VSP mutant at all depolarized potentials. Depleting endoplasmic reticulum Ca 2+ did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP 3 /Ca 2+ were not responsible. This study demonstrates for the first time that depleting PIP 2 per se inhibits NBCe1 activity. The data in conjunction with previous findings implicate a dual PIP 2 regulatory pathway for NBCe1 involving both PIP 2 itself and generated InsP 3 /Ca 2+ .